Treatment with immunosuppressive drugs is widely accepted as an effective treatment for bone marrow and solid organ transplantation to improve the graft survival. However, chronic rejection of transplants still has a considerable impact on the long term outcome. Moreover, many immunosuppressive drugs nonspecifically target the immune response, leading to unwanted side effects, such as weakened overall immune system. Thus, the goal in transplantation is the induction of a sustained state of specific tolerance to donor alloantigens with minimization or complete withdrawal of global immunosuppression.
CD4+CD25+Foxp3+ regulatory T cells (Treg) are negative regulators of immune responses to self- and foreign-antigens and play a critical role in maintaining immune tolerance by suppressing pathological immune responses in autoimmune diseases, transplant allograft rejection, and graft-versus-host-disease (GVHD).1-3 Upon adoptive transfer in rodents, Treg were found to control experimental autoimmune diseases,4 inhibit GVHD5,6 and prevent transplant allograft rejection,7,8 indicating that Treg-based therapy has a great therapeutic potential for these diseases in humans.
An important obstacle to Treg-based therapy has been the limited numbers of these cells that are available, as only about 1-2% of circulating human CD4+ T cells are Treg. Several groups have developed protocols to expand a large number of polyclonal CD4+CD25+ Treg in vitro with repeated stimulation by either CD3 and CD28 mAbs or artificial antigen-presenting cells (APC) for activation through CD3 and CD28, together with exogenous high-dose IL-2.9-11 polyclonal Treg may cause global immune suppression.4,7 In addition, since there are only few antigen-specific Treg in the population of the polyclonal Treg, very large numbers of non-specifically expanded Treg are required to inhibit bone-marrow allograft rejection in animal models.12 All of these characteristics of polyclonal Treg hamper their clinical applications.
In contrast, adoptive transfer of antigen-specific Treg has been shown to prevent and treat T-cell-mediated inflammatory diseases with high efficiency. In animal models, small number of antigen-specific Treg can suppress experimental autoimmune diseases,13 prevent GVHD and allograft rejection in bone marrow and solid organ transplantation.14,15 Importantly, the transfer of antigen-specific Treg prevented target antigen-mediated T-cell responses such as GVHD and allograft rejection but did not compromise host general immunity including the graft-versus-tumor activity and antiviral immunity.5,15-17 Based on these studies, antigen-specific Treg has substantial promise for human immunotherapy.
The reliable induction and expansion of rare antigen-specific Treg is technically challenging. Currently, several protocols for murine antigen-specific Treg induction and expansion have been reported in which either purified CD4+CD25− or CD4+CD25+ cells were co-cultured with autologous dendritic cells (DCs) pulsed with alloantigen in the presence of high-dose IL-2 or directly co-cultured with allogeneic DCs.14,18-20 Similar protocol has also been reported for generation of human antigen-specific Treg recently.21 In this protocol, antigen-specific CD4+CD25+ Treg can be generated by using the co-culture of CD4+CD25− T cells with allogeneic monocyte-derived DCs. However, the large-scale in vitro expansion of alloantigen-specific Treg is difficult because of certain features of DCs. For example, DCs are relatively rare in peripheral blood and are usually derived from apheresis or marrow sources including monocytes.22,23 Further, DCs are not homogeneous and include multiple subsets with different functional capacities.24 Finally, there is no effective way to expand human DCs so far.25 In addition, the current approaches to generate human DCs in vitro are expensive and laborious.26 
Schultze et al. reported a simple and low-cost method to expand large number human CD40-activated B cells up to 105,6-fold from human peripheral blood mononuclear cells (PBMC).27 These expanded B cells are effective as APCs and can efficiently induce antigen-specific T cells and cytotoxic T lymphocytes.26,27 
However, the art lacks an effective means of generating human antigen-specific Treg on a large scale. Thus, there exists a need in the art for a method of inducing or generating human antigen-specific Treg on a large scale.